Antigen presentation by Toxoplasma gondii-infected cells to CD4+ proliferative T cells and CD8+ cytotoxic cells

Cytotoxic cells specific for Toxoplasma gondii-infected cells were detected in the peripheral blood leukocytes from a patient with acute toxoplasmosis. The cytotoxicity was mediated by CD5+, CD4-, CD8+ cells. The cytotoxic T cells lysed Toxoplasma-infected target cells with HLA class I restriction. Two types of T cell clones were established from peripheral blood leukocytes of a patient with chronic toxoplasmosis; one was a CD5+, CD4-, CD8+ cytotoxic cell specific for Toxoplasma-infected cells, and the other was a CD5+, CD4+, CD8- proliferative cell that responded to Toxoplasma antigen. Toxoplasma-infected cell-specific cytotoxic cloned T cells recognize the infected target cells in the context of the HLA class I molecules, and the CD8 molecule was involved in the cytotoxicity. Toxoplasma antigen-specific proliferative cloned T cells were stimulated by Toxoplasma antigen-pulsed or Toxoplasma-infected cells in conjunction with HLA-DR molecule on the target cells. Thus, antigen presentation by Toxoplasma-infected cells for activation of both cytotoxic and proliferative T cells has been demonstrated.     

Macrophage Polarization Reflects T Cell Composition of Tumor Microenvironment in Pediatric Classical Hodgkin Lymphoma and Has Impact on Survival

Macrophages have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL) and have been suggested to have a negative impact on outcome. Most studies addressing the role of macrophages in cHL have relied on identification of macrophages by generic macrophage antigens, e.g., CD68. We have therefore conducted an in situ analysis of macrophage polarization in a series of 100 pediatric cHL (pcHL) cases using double staining immunohistochemistry, combining CD68 or CD163 with pSTAT1 (M1-like) or CMAF (M2-like). M1- or M2-polarised microenvironment was defined by an excess of one population over the other (>1.5). Expression of STAT1 and LYZ genes was also evaluated by RT-qPCR. Patients <14 years and EBV+ cases displayed higher numbers of CD68+pSTAT1+ cells than older children and EBV- cases, respectively (P=0.01 and P=0.02). A cytotoxic tumor microenvironment, defined by a CD8+/FOXP3+ ratio >1.5 was associated with higher numbers of CD68+pSTAT1+ (P=0.025) and CD163+pSTAT1+ macrophages (P<0.0005). Levels of STAT1 and LYZ expression were associated with the numbers of CD68+pSTAT1+ macrophages. EBV+ cHL cases disclosed a predominant M1 polarized microenvironment similar to Th1 mediated inflammatory disorders, while EBV- cHL showed a predominant M2 polarized microenvironment closer to Th2 mediated inflammatory diseases. Better overall-survival (OS) was observed in cases with higher numbers of CD163+pSTAT1+ macrophages (P=0.02) while larger numbers of CD163+CMAF+ macrophages were associated with worse progression-free survival (PFS) (P=0.02). Predominant M1-like polarization as disclosed by CD163+pSTAT1+/CD163+CMAF+ ratio > 1.5 was associated with better OS (P= 0.037). In conclusion, macrophage polarization in pcHL correlates with prevalent local T cell response and may be influenced by the EBV-status of neoplastic cells. Besides, M1-like and M2-like macrophages displayed differential effects on outcome in pcHL.     

Prognostic Implication of M2 Macrophages Are Determined by the Proportional Balance of Tumor Associated Macrophages and Tumor Infiltrating Lymphocytes in Microsatellite-Unstable Gastric Carcinoma

Tumor associated macrophages are major inflammatory cells that play an important role in the tumor microenvironment. In this study, we investigated the prognostic significance of tumor associated macrophages (TAMs) in MSI-high gastric cancers using immunohistochemistry. CD68 and CD163 were used as markers for total infiltrating macrophages and M2-polarized macrophages, respectively. The density of CD68+ or CD163+ TAMs in four different areas (epithelial and stromal compartments of both the tumor center and invasive front) were analyzed in 143 cases of MSI-high advanced gastric cancers using a computerized image analysis system. Gastric cancers were scored as “0” or “1” in each area when the density of CD68+ and CD163+ TAMs was below or above the median value. Low density of CD68+ or CD163+ macrophages in four combined areas was closely associated with more frequent low-grade histology and the intestinal type tumor of the Lauren classification. In survival analysis, the low density of CD163+ TAMs was significantly associated with poor disease-free survival. In multivariate survival analysis, CD163+ TAMs in four combined areas, stromal and epithelial compartments of both tumor center and invasive front were independent prognostic indicator in MSI-high gastric cancers. In addition, the density of CD163+ TAMs correlated with tumor infiltrating lymphocytes (TILs). Our results indicate that the high density of CD163+ TAMs is an independent prognostic marker heralding prolonged disease-free survival and that the prognostic implication of CD163+ TAMs might be determined by the proportional balance of TAMs and TILs in MSI-high gastric cancers.     

Abnormal thymocyte development and production of autoreactive T cells in T cell receptor transgenic autoimmune mice.

Development of a C57BL/6-+/+ TCR transgenic mouse containing the rearranged TCR alpha- and beta-chain specific for the Db + HY male Ag results in production of a nearly monoclonal population of early thymocytes expressing the Db + HY reactive TCR. These thymocytes are autoreactive in H-2Db male mice and undergo clonal deletion and down-regulation of CD8. To study the effect of the lpr gene on development of autoreactive T cells, these transgenic mice were backcrossed with C57BL/6-lpr/lpr mice. T cell populations in the thymus and spleen were analyzed by three-color flow cytometry for expression of CD4, CD8, and TCR. The thymus of TCR transgenic H-2b/b lpr/lpr male mice had an increase in percent and absolute number of CD8dull thymocytes compared to TCR transgenic H-2b/b +/+ male mice. However, there was not a complete defect in clonal deletion, because clonal deletion and down-regulation of CD8 was apparent in both +/+ and lpr/lpr H-2Db HY+ male mice compared to H-2Db HY- female mice. The phenotype of splenic T cells was almost identical in TCR transgenic +/+ and lpr/lpr males with about 50% CD4-CD8- T cells and 50% CD8+ T cells. However, there was a dramatic increase in the SMLR proliferative response of splenic T cells from TCR transgenic lpr/lpr males compared to TCR transgenic +/+ males. To determine the specificity of this response, spleen cells from TCR transgenic lpr/lpr and +/+ mice were cultured with irradiated H-2b/b and H-2k/k male and female spleen cells. T cells from TCR transgenic C57BL/6-lpr/lpr male mice had an increased proliferative response to H-2b/b male spleen cells compared to T cells from TCR transgenic C57BL/6(-)+/+ male mice, but both lpr/lpr and +/+ mice had a minimal response to irradiated H-2b/b female or H-2k/k male or female stimulator cells. The splenic T cells from TCR transgenic lpr/lpr mice also had an increased specific cytotoxic activity against H-2b/b male target cells compared to TCR transgenic +/+ mice. These results demonstrate that there is a defect in negative selection of self-reactive T cells in the thymus of lpr/lpr mice and a defect in induction or maintenance of clonal anergy of self-reactive T cells in the periphery of lpr/lpr mice.     

Expression and function of a 90-kilodalton homodimeric molecule (10D1 antigen) on human thymocytes

The 10D1 Ag is a 90-kDa homodimeric molecule specifically expressed on a subpopulation of human T cells, and is involved in an alternative pathway of T cell activation. In the present study, we have examined the expression and function of the 10D1 Ag on human thymocytes. Three-color FMF analysis showed that the 10D1 Ag was highly expressed on minor but distinct subpopulations of double-negative and CD4 single-positive thymocytes, and weakly on a part of double-positive thymocytes, but not on CD8 single-positive thymocytes. In double-negative thymocytes, the vast majority of 10D1+ cells were immature thymocytes of CD7+2+3- phenotype. Interestingly, 10D1 mAb could induce the proliferation of CD4 single-positive thymocytes in the presence of goat anti-mouse Ig to cross-link the 10D1 Ag. The treatment of thymocytes with OKT4 mAb plus C but not with OKT8 mAb plus C totally abrogated the proliferative response induced by 10D1 mAb, indicating that the 10D1-responsible thymocytes were of CD4+8- phenotype. This 10D1 mAb-induced thymocyte proliferation was perfectly dependent on the endogenous IL-2/IL-2R system since a complete inhibition was observed with anti-IL-2 and anti-IL-2R mAb. The proliferating CD4 single positive thymocytes predominantly expressed the IL-2R alpha (p55) but not a detectable level of the IL-2R beta (p75). These results indicate that, although the 10D1 Ag can be detected on the CD7+2+3-4-8- thymocytes, its functional expression is restricted to a minor more mature CD4+ thymocyte population as well as in peripheral blood T cells, and the implications of these findings are discussed.     

Quantitative changes in characteristics of immunity in longlivers

In 93 persons aged 90 to 103 years old the subpopulation compositon of lymphocytes and the level of thymic serum activity (TSA) was studied. In the presence of polymorbidity the low level of TSA was observed in 81% of examinees, with the quantitative deficiency in the relative and absolute number of CD3+CD8+ T-lymphocytes (62% and 78% respectively) and the excess in the number CD3+CD4-CD8- T-lymphocytes (42% and 57% respectively). Among CD3+CD4-CD8- T-lymphocytes cells with phenotypes CD3+/-CD4-CD8-CD16- and CD3+CD4-CD16+ were determined. In prolonged imunomonitoring the reciprocacity of changes in the amount of CD3+/-CD4-CD8-CD16- T-lymphocytes was established in relation to the level of TCA and the number of CD3+CD8+ T-lymphocytes. The reversibility of these changes is indicative of the potential of reactivity of the immune system in longlivers. The positive result of laser therapy in persons with quantitative deficiency of CD3+CD8+ T-lymphocytes and the presence of cells with phenotype CD3+/-CD4-CD8-CD16- was associated with the restoration of the size of CD3+CD8+ subpopulation and a decrease in an amount of CD3+/-CD4-CD8-CD16-. The unfavorable prognostic sign was, seemingly, the quantitative deficiency of CD3+CD8+ T-lymphocytes with the low level of TCA in the absence of CD3+/-CD4-CD8-CD16- T-lymphocytes.     

The FOXP3+ subset of human CD4+CD8+ thymocytes is immature and subject to intrathymic selection

FOXP3, believed to be the regulatory T (Treg)-cell determining factor, is already expressed at the CD4+CD8+ thymocyte stage, but there is disagreement whether these cells are the precursors of mature CD4+CD8(-) Treg cells. Here, we provide a quantitative analysis of FOXP3 expression in the human thymus. We show that a subset of CD4+CD8+ cells already expressed as much FOXP3 as the FOXP3+ CD4+CD8(-) cells, and like mature Treg cells were CD127 low. In contrast to earlier data, CD8+CD4(-) thymocytes expressed significantly lower levels of FOXP3 than either the CD4+CD8+ or CD4+CD8(-) subsets. The CD4+CD8+ double-positive cells also expressed recombination-activating gene-2, suggesting that they were still immature. Although the FOXP3+ double-positive cells are thus putatively the precursors of the mature CD4+CD8(-)FOXP3+ subset, their frequency did not predict the frequency of more mature Treg cells, and analysis of T-cell antigen receptor repertoire showed clear differences between the two subsets. Although these data do not rule out an independent CD4+CD8+ Treg cell subset, they are consistent with a model of human Treg cell development in which the upregulation of FOXP3 is an early event, but the first FOXP3+ population is still immature and subject to further selection. The upregulation of FOXP3 may thus not be the final determining factor in the commitment of human thymocytes to the Treg cell lineage.     

Disorders in the immune responses of T- and B-cells in mice administered intravenous verotoxin 2

To obtain a better insight into the pathogenesis of verotoxin-producing Escherichia coli (VTEC)-associated diseases, we explored the effect of verotoxin 2 (VT2) on the immune response in mice. The distribution of lymphocyte phenotypes and the lymphocyte immune response were examined after intravenous administration of VT2 to mice. Among the peripheral lymphocytes and splenocytes of 4-week-old C57BL/6 mice, there was first of all a decrease in T-cells, which began 24 h after intravenous administration of VT2 (50 ng/kg, lethal dose). The CD4+ cell subpopulations of the peripheral blood and spleen were significantly decreased at 24 h, while the B220+ splenocyte subpopulation was markedly decreased at 45 h after VT2 administration. In the thymus, a decrease in CD4+CD8+ cells was predominantly observed near death. Interestingly, in E. coli lipopolysaccharide (LPS)-responder mouse strains (C57BL/6 and C3H/HeN) cotreated with LPS, the susceptibility to VT2 was enhanced, and the increase in B220+ cells induced by LPS alone was suppressed. Furthermore, splenocytes from C57BL/6 mice treated with VT2 (50 ng/kg) 6-24 h earlier reduced LPS-induced proliferative responses to 50-52% of that in control cells, indicating that the effect of VT2 on the immunoresponse seen in vivo may be negatively exerted on the proliferation of the cells. In addition, the number of splenocytes that produced anti-sheep red blood cell antibody was decreased in mice treated with VT2. These results suggest that VTEC infection may eliminate CD4+ and CD8+ T-cells and B-cells by affecting their survival and proliferative responses, leading to reduced antibody production.     

Characterization of monocyte/macrophage subsets in the skin and peripheral blood derived from patients with systemic sclerosis

Introduction

Recent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.

Methods

Skin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.

Results

In the skin from SSc patients, the number of CD163⁺ cells or CD204⁺ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14⁺ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14⁺ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163⁺ cells belong to CD14^brightCD204⁺ population.

Conclusions

This is the first report indicating CD163⁺ or CD204⁺ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14^brightCD163⁺CD204⁺ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163⁺ or CD204⁺ macrophages in the skin.     

Preferential accumulation of mature NK cells during human immunosenescence.

The major histocompatibility complex-unrestricted, cell-mediated, constitutive anti-tumor cytotoxic function of natural killer cells is highly preserved in healthy elderly. A study of the dynamics of expression of natural killer cell-associated phenotypes during immunosenescence shows that selective, bidirectional, and disproportionate changes in certain natural killer cell subset number and ratio take place during aging. The mean natural killer cell subset ratio (%CD16+CD57+ over %CD56+CD57-) gradually increases from a young adult level of 0.7 to 4.6 with advancing age predominantly due to a tripling of %CD16+57+ cells as opposed to a moderate decrease (-54%) in %CD56+57- phenotype. The parallel increase in natural killer phenotype ratio and cytotoxic activity might represent a shift in the maturity status of these cells. Based on these findings, a model of natural killer cell immunosenescence is proposed. It is concluded that not all immunosenescent changes need be detrimental; some may even improve the potential for survival and represent an adaptational immunosenescent change.     

Human immunodeficiency virus type 1 (HIV-1)-specific CD4+ T cells that proliferate in vitro detected in samples from most viremic subjects and inversely associated with plasma HIV-1 levels

Diminished in vitro proliferation of human immunodeficiency virus type 1 (HIV-1)-specific CD4+T cells has been associated with HIV-1 viremia and declining CD4+ T-cell counts during chronic infection. To better understand this phenomenon, we examined whether HIV-1 Gag p24 antigen-induced CD4+ T-cell proliferation might recover in vitro in a group of subjects with chronic HIV-1 viremia and no history of antiretroviral therapy (ART). We found that depletion of CD8+ cells from peripheral blood mononuclear cells (PBMC) before antigen stimulation was associated with a 6.5-fold increase in the median p24-induced CD4+ T-cell proliferative response and a 57% increase in the number of subjects with positive responses. These p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC were associated with expansion of the numbers of p24-specific, gamma interferon (IFN-gamma)-producing CD4+ T cells. Among the 20 viremic, treatment-naive subjects studied, the only 5 subjects lacking proliferation-competent, p24-specific CD4+ T-cell responses from CD8-depleted PBMC showed plasma HIV-1 RNA levels > 100,000 copies/ml. Furthermore, both the magnitude of p24-induced CD4+ T-cell proliferative responses from CD8-depleted PBMC and the frequency of p24-specific, IFN-gamma-producing CD4+ T cells expanded from CD8-depleted PBMC were associated inversely with plasma HIV-1 RNA levels. Therefore, proliferation-competent, HIV-1-specific CD4+ T cells that might help control HIV-1 disease may persist during chronic, progressive HIV-1 disease except at very high levels of in vivo HIV-1 replication.     

Apoptosis resistance in pigmented villonodular synovitis

OBJECTIVE: Pigmented villonodular synovitis (PVNS) is a proliferative lesion originating from synovial tissue with a locally aggressive behaviour. We analysed the pathogenetic role of apoptosis resistance for sustained cell proliferation in PVNS. METHODS: The expression of bcl-2, p53 and Ki-67 was examined in 80 cases of PVNS using immunohistochemistry. In 43 of these cases, DNA content and distribution of cell-cycle phases were investigated by flow cytometry. Additionally, 10 cases of PVNS were analysed by multi-parametric flow cytometry for expression of p53, caspase3, and bcl-2 and by TUNEL to detect DNA fragmentation. RESULTS: No apoptotic cell fractions were detected in any investigated cases. Expression of bcl-2 was found in 84% of cases (up to 6.5% of cells) and was significantly associated with DNA-fragmentation observed by TUNEL (p=0.037). Orthologous p53 expression was observed in 37% of cases. The level of p53 expression correlated with the proliferative activity and the expression of both caspase3 (p=0.017) and bcl-2 (p=0.0013). (No statistically significant correlations between expression of bcl-2, p53, caspase3, DNA fragmentation or proliferative index and age, sex of patients, disease recurrence, growth pattern or size of lesion were found). CONCLUSION: Apoptosis resistance is a critical event in the progression of PVNS and may contribute to the survival of the proliferating synovial cells in PVNS and to the permanent slow progression of these lesions.     

Analysis of the human neonatal thymus: evidence for a transient thymic involution

The neonatal period is marked by the impairment of the major components of both innate and adaptive immunity. We report a severe depletion of cortical CD4+CD8+ double-positive thymocytes in the human neonatal thymus. This drastic reduction in immature double-positive cells, largely provoked by an increased rate of cell death, could be observed as early as 1 day after birth, delaying the recovery of the normal proportion of this thymocyte subset until the end of the first month of postnatal life. Serum cortisol levels were not increased in newborn donors, indicating that the neonatal thymic involution is a physiological rather than a stress-associated pathological event occurring in the perinatal period. Newborn thymuses also showed increased proportions of both primitive CD34+CD1- precursor cells and mature TCRalphabetahighCD69-CD1-CD45RO+/RAdull and CD45ROdull/RA+ cells, which presumably correspond to recirculating T lymphocytes into the thymus. A notable reinforcement of the subcapsular epithelial cell layer as well as an increase in the intralobular extracellular matrix network accompanied modifications in the thymocyte population. Additionally neonatal thymic dendritic cells were found to be more effective than dendritic cells isolated from children's thymuses at stimulating proliferative responses in allogeneic T cells. All these findings can account for several alterations affecting the peripheral pool of T lymphocytes in the perinatal period.     

Immunohistochemical characterization of the cellular infiltrate in Jorge Lobo's disease

Few studies have been conducted to evaluate the cellular composition of the granulomatous lesions induced by Lacazia loboi. Thus, the objective of the present study was to characterize the mononuclear cell population present in cutaneous lesions obtained from 15 patients with Jorge Lobo's disease. Histological sections were stained with hematoxylin-eosin and methenamine silver and the following mononuclear cells were identified by immunohistochemistry: T lymphocytes (CD3+), helper T lymphocytes (CD4+), cytotoxic T lymphocytes (CD8+), B lymphocytes (CD20+), plasma cells (CD79+), natural killer cells (CD57+) and histiocytes (CD68+). This study showed that the inflammatory infiltrate mainly consists of histiocytes and multinucleated giant cells, in addition to the presence of a large number of fungal cells. The identified inflammatory cells showed the following frequency: CD68+ histiocytes > CD3+ T lymphocytes > CD4+ T > CD8+ T lymphocytes > CD57+ natural killer cells > CD79+ plasma cells > CD20+ B lymphocytes. Based on the findings of a large number of fungal cells in the infected tissues and the disorganized cell arrangement in the granuloma, we hypothesize that patients with Jorge Lobo's disease present immunoregulatory disturbances, which are likely to be specific and perhaps responsible for the lack of containment of the pathogen.     

Myelin antigen-specific CD8+ T cells are encephalitogenic and produce severe disease in C57BL/6 mice

Encephalitogenic T cells that mediate experimental autoimmune encephalomyelitis (EAE) are commonly assumed to be exclusively CD4+, but formal proof is still lacking. In this study, we report that synthetic peptides 35-55 from myelin oligodendrocyte glycoprotein (pMOG(35-55)) consistently activate a high proportion of CD8+ alphabetaTCR+ T cells that are encephalitogenic in C57BL/6 (B6) mice. The encephalitogenic potential of CD8+ MOG-specific T cells was established by adoptive transfer of CD8-enriched MOG-specific T cells. These cells induced a much more severe and permanent disease than disease actively induced by immunization with pMOG(35-55). CNS lesions in pMOG(35-55) CD8+ T cell-induced EAE were progressive and more destructive. The CD8+ T cells were strongly pathogenic in syngeneic B6 and RAG-1(-/-) mice, but not in isogeneic beta2-microglobulin-deficient mice. MOG-specific CD8+ T cells could be repeatedly reisolated for up to 287 days from recipient B6 or RAG-1(-/-) mice in which disease was induced adoptively with <1 x 10(6) T cells sensitized to pMOG(35-55). It is postulated that MOG induces a relapsing and/or progressive pattern of EAE by eliciting a T cell response dominated by CD8+ autoreactive T cells. Such cells appear to have an enhanced tissue-damaging effect and persist in the animal for long periods.     

Redistribution of natural killer (NK) cell frequency and NK cytotoxic activity in primary IgA nephropathy.

Recent findings have indicated an imbalance of immune responsiveness in primary IgA nephropathy (IgAN). Thus natural killer (NK) cell frequency and NK cytotoxicity were evaluated in fifteen IgAN patients. CD8+, CD11+, CD56+ and CD57+ lymphocyte percentages in IgAN individuals fell within normal values, while a significant decrease of CD16+ cells was observed in the same group of patients. In contrast, NK activity overlapped that seen in controls as assessed by an agarose-single cell cytotoxic assay. To further investigate the discrepancy between CD16+ cell level and NK cytotoxic activity in IgAN, the proportion of CD11+ CD57+, CD56+ CD16+ and CD57+ CD16+ lymphocytes was determined. In spite of the unaffected CD56+ CD16+ cell frequency, IgAN subjects exhibited a significant decrease of CD11+ CD57+ and CD57+ CD16+ lymphocyte percentages in comparison to controls. It is suggested that a redistribution of NK lymphocyte subsets occurs in IgAN. This may have an important role in the impairment of the immunoregulatory network.